As I finish pyrosequencing, I get the following error messages sometime.
(I use Pyromark Q96 ID (From Qiagen) pyrosequencer to quantify percentage of CpG islands' methylation in the promoter region)
General warning:
Failed reference sequence pattern (Overall)
CpG warning:
1. Uncertain due to high CpG pattern deviation.
2. Uncertain due to high CpG sum deviation.
Can anyone please explain what these warnings mean? Can I bypass these warnings without affecting the ingenuity of my results.
Also, I have found that sometime the overall methylation percentage is equal between the exact promoter sequence treated differently, but there is a variation among individual CpG islands (For example: the overall methylation of 8 CpG islands in two samples (sample A and Sample B) of C-kit promoter are 35% for both the samples, however, there is variation in methylation percentage for CpG 1 CpG2, CpG3........CpG8 of Sample A and B). Is this a normal phenomenon?
Thanks in Advance.
-Prabin
this link is useful
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4775062/
regards
Dear Prabin,
"Failed reference sequence pattern" error message - might be related to the fact, that in your pyrogram there is some signal at the position where there should be no signal/nucleotide.
And there are variety reasons why it might happen.
But in general, if you want to look what every specific error message means, you can look it up in the Pyrosequencing manual.
The manual should be available at the Qiagen web-site.
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